Project Summary Tuberculosis (TB), an infectious disease caused by species of Mycobacterium tuberculosis complex (MTC), remains one of the major public health problems worldwide, with nine million new cases and about 1.5 million deaths each year. In addition, improper use of antibiotics in chemotherapy of drug-susceptible TB patients results in anti-TB drug resistance, which complicates TB care and control. About 95% of lethal cases occur in resource-limited high-burden countries, where there is a lack of diagnostic capacity. Undiagnosed or improperly diagnosed patients develop more severe disease and remain a source of infection for the wider public. At the same time, efficient point-of-care (POC) TB diagnostics in low resource settings can improve health outcomes by facilitating more effective treatment, reducing the patient dropout rate and limiting spread of the infection. The tests of such format are lacking for drug susceptibility testing (DST) of TB. Therefore, new diagnostic tools that are simple enough to be used ?in the field? and still be able to correctly diagnose for TB and identify drug-resistance are needed. The goal of this high-risk, high-reward exploratory project is to take advantage of the recent advancements in DNA nanotechnology and molecular sensing to significantly improve diagnostics capabilities for TB. We propose to develop POC-compatible highly selective sensors based on cascades of deoxyribozymes with visual signal readout for detection of active pulmonary TB and testing for mutations conferring multidrug resistance (MDR). Visual deoxyribozyme cascade sensors will be able to produce highly reliable results within the period of usual doctor's visit, and be able to detect less than 104 cells of MTC species without the need for expensive equipment. We will design cascade sensors targeting MTC rRNA and fragments of the genes containing point mutations conferring resistance to the most potent first-line anti-TB drugs rifampicin and isoniazid. We will optimize the assay using isolated bacterial nucleic acids, whole bacterial cells and bacteria-spiked human sputum samples. We will compare the assay with acid fast smear microscopy and culture-based tests, which are currently used for TB detection and DST in the resource-limited settings. The technology proposed here is promising for diagnostics of TB, as well as other pathogens, particularly in resource-constrained high-burden countries, which may revolutionize healthcare worldwide.